Protein interactions in solution: Implications for protein aggregation and separation.

摘要

Protein aggregation is ubiquitous in production and formulation of therapeutic proteins and is the probable cause of a number of neurodegenerative diseases. Protein intermolecular attractions are responsible for protein self-assembly and protein aggregation. Protein intermolecular interactions can be controlled by solution conditions during protein separation processes such as precipitation and crystallization. A more complete understanding of protein intermolecular forces will help to elucidate the mechanism of protein aggregation; it will also contribute toward establishing rational design criteria for selecting suitable solution conditions during recombinant protein production.; In this work, we have studied the mechanism of protein aggregation and protein interactions under conditions corresponding to protein-production processes. Aggregated proteins have structures that vary from amorphous to highly ordered beta-sheet aggregates. One of the most important examples of a highly ordered aggregate is the insoluble amyloid fibril that has been found to be associated with several neurodegenerative diseases. In Chapter 2, we have been able to convert a polypeptide derived from the beta-sheet region of T4 lysozyme into amyloid fibrils. Peptide fibril formation is facilitated by a moderate content of alpha-helix in the initial peptide solution. A stable alpha-helix inhibits fibril formation. These results support the view that amyloid fibrillogenesis is a common generic property of all proteins and polypeptides.; Molecular chaperones can prevent cellular protein misfolding and aggregation by temporarily binding to newly synthesized polypeptides or unfolded proteins. DnaK is the Hsp70 (70 kDa heat-shock protein) molecular chaperone of Escherichia coli. It binds preferably with a peptide that consists of a hydrophobic core and flanking regions enriched in basic residues. In Chapter 3, we have delineated the electrostatic contributions to the binding free energy between molecular chaperones and peptides. We have also developed an approximate analytic model for prediction of the electrostatic contribution to the potential of mean force for a pair of dissimilar dipolar particles. The calculated electrostatic free energy of binding shows reasonable agreement with that obtained from fluorescence-anisotropy measurements.; Many protein separation processes, including protein precipitation and crystallization, are intimately related to control of protein-protein interactions. The strength of a protein-protein interaction can be described by the osmotic second virial coefficient, B22. Solvents used in protein precipitation and crystallization are often aqueous mixtures of buffer salts with organic solvents such as polymers, alcohols and polyols. In Chapter 4, we have measured B22 of hen-egg lysozyme in salt solution with several alcohol additives. All the alcohols used in this study raise the second virial coefficient, indicating stronger protein-protein repulsion. We describe the alcohol effect using a potential of mean force (PMF) model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions.; In Chapter 5, we have measured B22 for partially unfolded lysozyme in the presence of GdnHCl at several concentrations. Lysozyme inter-particle interactions are least repulsive and hydrodynamic interactions are least attractive at intermediate (1∼2M) GdnHCl concentrations.; The work described in this thesis has improved our understanding of interactions between protein molecules. Such understanding is of vital importance in understanding protein function in nature and in protein processing biotechnology.